© 1991 Oxford University Press
Nucleic Acids Research, Vol. 19, No. 11 2929
Measurement of the sequence specificity of covalent DNA
modification by antineoplastic agents using Taq DNA
polymerase
Mauro Ponti 12 , Stephen M.Forrow1, Robert L.Souhami1, Maurizio D'lncalci2 and
John A.Hartley 1 *
department of Oncology, University College and Middlesex School of Medicine, 91 Riding House
Street, London W1P 8BT, UK and 2lstituto di Ricerche Farmacologiche 'Mario Negri', via Eritrea 62,
20157 Milan, Italy
Received March 5, 1991; Revised and Accepted April 29, 1991
ABSTRACT
A polymerase stop assay has been developed to
determine the DNA nucleotide sequence specificity of
covalent modification by antineoplastic agents using
the thermostable ONA polymerase from Thermus
aquaticus and synthetic labelled primers. The products
of linear amplification are run on sequencing gels to
reveal the sites of covalent drug binding. The method
has been studied in detail for a number of agents
including nitrogen mustards, platinum analogues and
mitomycin C, and the sequence specificities obtained
accord with those obtained by other procedures. The
assay is advantageous in that it is not limited to a single
type of DNA lesion (as in the piperidine cleavage assay
for guanine-N7 alkylation), does not require a strand
breakage step, and is more sensitive than other primer
extension procedures which have only one cycle of
polymerization. In particular the method has
considerable potential for examining the sequence
selectivity of damage and repair in single copy gene
sequences in genomic DNA from cells.
INTRODUCTION
Sequence selective reactions with DNA have been demonstrated
for several cancer chemotherapeutic agents including bleomycin
(1), mitomycin C (2), cis-dichlorodiammine platinum (II)
(cisplatin, 3), chloroethylnitrosoureas (4) and nitrogen mustards
(5-7). In the latter two classes the relative extent of alkylation
of guanine-N7 positions, the major site of DNA reaction with
such compounds, was determined using a modification of the
Maxam and Gilbert method of DNA sequence determination (8)
in which sites of alkylation in isolated DNA are quantitatively
converted to strand breaks with hot piperidine (9). Most nitrogen
mustards were found to have similar patterns of alkylation, with
sites of greatest reactivity being at runs of consecutive guanines
although the substituent attached to the reactive group on the drug
* To whom correspondence should be addressed
could introduce a distinct sequence preference as demonstrated
for uracil and quinacrine mustards (5-7).
The piperidine cleavage assay is however limited to the
detection of a single type of DNA lesion. Various kinds of DNA
damage block the 3' to 5' exonuclease action of enzymes such
as E.coli exonuclease III or the 5' to 3' exonuclease action of
lambda exonuclease, the latter enzyme appearing to be
particularly sensitive to obstructions in the minor groove of DNA
and not to alkylation of guanine-N7 in the major groove (10).
Cisplatin forms an adduct at guanine-N7 which is not revealed
by the piperidine cleavage method but produces blocks to
exonuclease HI (3), and, using a primer extension procedure with
the Klenow fragment of DNA polymerase I, it has been shown
that this drug forms adducts that inhibit DNA synthesis in vitro
at all (dG)n (n> 2) sequences (11). More recently, the primary
blocking lesions for the same enzyme formed by cisplatin on
double-stranded DNA were found to be identical to those
produced by the chloroethylnitrosoureas (12). None of these
methods, however, are suitable for examining the sequence
selectivity of damage in vivo.
We now report a new method of detecting the sites of covalent
binding to DNA by various antineoplastic agents with a primer
extension procedure utilizing multiple cycles of polymerization
with the thermostable DNA polymerase from Thermus aquatius.
The results obtained are compared with the sequence selectivities
previously obtained by other methods. The method should be
applicable to the measurement of the sequence selectivity of
damage and repair in single copy gene sequences in genomic
DNA.
MATERIALS AND METHODS
Materials
Reagents were obtained from the following sources:
mechlorethamine, cis-dichlorodiammine platinum (11) and
mitomycin C, Sigma Chemical Company; carboplatin, Bristol
2930 Nucleic Acids Research, Vol. 19, No. 11
Myers; quinacrine mustard, Fluka Chemical Company; uracil
mustard, Upjohn Company; melphalan, Wellcome Foundation;
pBR322 DNA, T4 polynucleotide kinase and restriction
endonucleases, Northumbria Biological Limited; (7-32p)ATP
(5000 Ci/mmol), Amersham; Amplitaq recombinant Taq DNA
polymerase, Perkin Elmer Cetus. All other reagents were of the
highest purity available.
Drugs (except melphalan) were dissolved in DMSO at 50mM
immediately prior to use and then further diluted in water. A
stock solution of melphalan was prepared at lOmM in 0.1M
hydrochloric acid and stored frozen.
Preparation of damaged DNA
pBR322 DNA was digested with Bam HI and Sal I restriction
enzymes and purified by standard phenol/chloroform extraction
and ethanol precipitation reactions (13). DNA (0.5/ig per sample)
was reacted with drug in 25mM triethanolamine, lmM EDTA,
pH7.2 buffer for 1 hour in a final volume of 50^1. DNA was
then precipitated twice with ethanol and vacuum dried. In the
case of mitomycin C reaction was performed in the presence of
a 3 times molar concentration of sodium dithionite in buffer
removed of oxygen by bubbling with nitrogen.
Primer extension
A synthetic oligonucleotide primer of the following sequence was
used: 5'-TATGCGACTCCTGCATTAGG-3'. The primer was
5'-end labelled with [7-32p] ATP using T4 polynucleotide
kinase. The linear amplification of DNA was carried out in a
total volume of 100/il containing 0.5^g DNA, 10/tl lOxbuffer
(670mM Tris pH 8.4, 20mM MgCl2), 0.25ng labelled primer,
250/tM of each dNTP and 1U Taq polymerase. After gentle
mixing the samples were overlaid with 2 drops of mineral oil
and then incubated in a thermal cycler.
The amplification procedure was carried out for 30 cycles, each
consisting of 1 min denaturation at 95 °C, 2 min annealing at 60°C
and 2 min chain elongation at 72°C. After the last cycle, samples
were chilled on ice, extracted with a mixture of chloroform/
woamylalcohol (24:1), precipitated with ethanol and dried. DNA
fragments were separated on 0.4mm, 6% polyacrylamide gels
containing 7M urea and a tris-boric acid -EDTA buffer system.
Running time was approximately 3h at 3000V, 55°C. Gels were
transferred to filter paper and dried. Following autoradiography
relative band intensities were determined by microdensitometry
with an LKB Ultrascan-XL laser densitometer.
Piperidine Cleavage Method for guanine-N7 alkylation
The BamHI-Sall fragment of pBR322 was 3'-singly end-labelled
at the Sail site by standard procedures and drug treated as
described above.
Salt-free DNA pellets were resuspended in freshly diluted 1M
piperidine and incubated at 90°C for 15 min to quantitatively
convert sites of guanine-N7 alkylation into strand breaks (9).
Samples were lyophilised, washed twice with distilled water then
resuspended in formamide loading buffer, heated at 90°C for
1 minute and chilled in an ice bath prior to loading onto
denaturing polyacrylamide gels as described above.
RESULTS
A synthetic 20 base oligonucleotide primer was synthesised
complimentary to bases 621-640 of the 273 base pair BamHISall fragment of pBR322 (bases 375-650). Following annealing
f 9 h
i
1
a b c d e
i
If *
.,---400
it
..-450 - '
...... G4-""
. —-G3
,-
r
•> m *
3 -
500 —-
n
G3
1
I
G
%+
II
•-
lilt
550
Figure 1. Autoradiograms of 6% denaturing sequencing gels showing the blocks
to Taq DNA polymerase produced by a number of cancer chemotherapeutic agents.
The left and right hand panels are from two separate experiments. Lanes a and
j are control, unmodified BamHI-Sall fragment of PBR322 DNA. Lanes b-i are
drug treated DNA fragments: lane b, 5jtM melphalan; lanes c and h, 2.5jiM
cisplatin; lane d, 0.5^M quanacrine mustard; lanes e and f, 20/tM
mechlorethamine; lane g, 2.5/iM uracil mustard; lane i, 250/JM carboplatin. The
base sequence position in pBR322 DNA is indicated, taking into account the
termination by Taq polymerase one nucleotide before a modified base, and the
positions of runs of 3 or more guanines also indicated. Arrows correspond to
the 5 sites of 5'-CGCC-3' within the sequence.
of the 5'end-labelled primer, extension with Taq DNA
polymerase produced a ftill length fragment of 263 base pairs
in length. Several cancer chemotherapeutic agents were examined
for their ability to block the progress of the polymerase and the
results for several agents are presented in figure 1 and the
corresponding densitometric traces in figure 2. The left and right
panels are from two separate experiments. In both cases control,
unmodified DNA (lanes a and j) show complete chain elongation
with very few sites of early termination. Interestingly, these sites
(accounting for < 1 % of total product) are different in the two
experiments, but are consistent with each experiment.
Figures 1 and 2, lanes b-i show the results from DNA that
has been modified with various nitrogen mustards or platinum
Nucleic Acids Research, Vol. 19, No. 11 2931
i
9 i
550
GCUGGTTGTC
550
500
450
base position |
i
450
500
AGGCGGCCGG
TGCCCGGACG
GTGGTATGGG
TGCGGCTTTG
TCCGCGGTO;
TTGGCGTGGA
CACCGCCJGCC
ACTACGCCCG
450
ACCCGCTATA
Figure 2. Densitomeric traces of the autoradiograms shown in figure 1, where traces a-j correspond to autoradiogram lanes a-j. The full base sequence in pBR322
is also indicated; as are the 5 sites of 5'-CGCC-3' within the sequence (arrows) in the uracil mustard trace (lane g).
analogues. In each case the level of modification is low, with
at most one modification/DNA fragment so that qualitative
comparisons can be made. In general all compounds showed a
preference for runs of guanines (of which runs of 3 or more are
indicated in figure 1) over isolated guanines. Of the nitrogen
mustards mechlorethamine (lanes e and f) and melphalan (lane
b) gave very similar patterns of alkylation, whereas quinacrine
(lane d) and uracil mustards (lane g) gave distinctly different
patterns. In particular quinacrine mustard (lane d) showed a strong
preference for 5'-GGPu-3' and 5'-GTPu-3' sites (where G is the
alkylated base and Pu= purine). This can be seen for example
at bases 546 and 518 which are 5'-GGPu-3' and 5'-GTPu-3' sites
respectively, and from the run of three guanine at bases 511 -513
in the sequence 5'-TGGGTA-3' where the two outer guanines
are heavily alkylated compared to the central guanine. Uracil
mustard (lane g) showed a reduced reactivity with runs of
guanines and a preference for some 5'-PyGC-3' sites
(Py=pyrimidine) and in particular 5'-CGCC-3' sequences (as
indicated by arrows in figures 1 and 2), which are weak sites
of alkylation by the other drugs.
Cisplatin (lanes c and h) and carboplatin (lane i) gave similar
patterns of blocking lesions which were almost exclusively at Gn
(n> 2) sequences. Approximately 100-fold more carboplatin was
required to give a similar extent of modification to cis-platin.
The reproducibility of the technique is indicated by the patterns
of blocking by mechlorethamine (lanes e and f) and cisplatin
(lanes c and h) in the two experiments shown in the left and right
panels of figures 1 and 2.
All the compounds shown in figure 1 react primarily with DNA
at the guanine-N7 position. In the case of the nitrogen mustards
550
500
base
position
450
|
Figure 3. a) Densitometric traces showing a comparison of the Taq polymerase
stop assay (upper trace) and the piperidine cleavage-based sequencing assay for
guanine-N7 alkylation (lower trace) for the antitumour agent mechlorethamine.
The data from two separate gels is aligned making allowance for the fact that
piperidine-cleaved DNA runs faster than primer-extended products of identical
sequence, b) Densitometric trace showing blocks to Taq polymerase by the
antitumour agent mitomycin C (100/iM) under reducing conditions.
2932 Nucleic Acids Research, Vol. 19, No. 11
treatment with hot piperidine cleaves the DNA at sites of guanine
N7 alkylations (9). Early experiments included such a cleavage
step prior to primer extension with Taq polymerase. This was
found not to be required, however, since identical results were
obtained with or without the piperidine cleavage step (data not
shown). A comparison of die standard piperidine cleavage
sequencing based assay for guanine-N7 alkylation with that of
the Taq polymerase primer extension assay on the same sequence
of DNA yielded similar results for the nitrogen mustards as
demonstrated in the case of mechlorethamine in figure 3 (a).
The potential of the method to study DNA damage at sites other
man at guanine-N7 in the major groove was assessed. For
example mitomycin C which is known to alkylate and cross-link
DNA under reducing conditions via the N2-amino group of
guanine in the minor groove (14), produced blocking lesions to
the Taq polymerase (figure 3(b)). Again with this agent a clear
preference is seen for runs of contiguous guanines.
DISCUSSION
A polymerase stop assay is presented for the determination of
the sequence specificity of covalent DNA modification by
antineoplastic agents using the thermostable DNA polymerase
from Thermus aquaticus and synthetic labelled primers. The
products of linear amplification are run on sequencing gels to
reveal the sites of covalent drug binding. The assay is
advantageous over other methods in that it is not limited to a
single type of DNA lesion (as in the piperidine cleavage assay
for guanine-N7 alkylation), does not require a strand breakage
step, and is more sensitive than other primer extension procedures
which have only one cycle of polymerization.
For the agents studied the sequence selectivities obtained
compare favourably with those obtained previously by other
methods. The nitrogen mustards have been studied in detail using
a sequencing based procedure (5—7) showing the general
preference for runs of guanines, and the unique reactivities for
uracil and quanicrine mustards observed in the present study.
Mechanisms to explain these selectivities have been proposed (6,
15). The selectivity of cisplatin also corresponds with the known
preference of this compound for GG sequences (16) and with
its ability to block E. Coli DNA polymerase I at all Gn (n > 2)
sites (11,12). Carboplatin gave the same pattern of reactivity as
cisplatin but at a 100-fold higher dose. This confirms previous
reports on the kinetics of interaction of the two compounds with
DNA, as predicted by their 100-fold differing rates of aquation
(17). Importantly the method is also capable of detecting
alkylations in the minor groove of DNA as demonstrated for
mitomycin C. Using a X exonuclease stop assay this agent has
recently been shown to covalently modify DNA preferentially
at guanine residues within 5'-GG and 5'-CG sequences (18).
The recently developed technique of in vivo footprinting of
protein-DNA interactions involves the treatment of whole cells
with dimethylsulphate (DMS), an alkylating agent which
methylates at the guanine-N7 position (19,20). Proteins bound
to DNA can alter the accessibility of DMS to guanines at or near
the binding site (21). After purification of DNA, both in vitro
and in vivo DMS-treated samples are quantitatively cleaved at
sites of guanine-N7 alkylation with piperidine and compared to
reveal footprints by primer extension assays (22,23). Piperidine,
however is known to inhibit Taq polymerase (23) and must be
removed completely. More recently a simplified method of in
vivo footprinting was proposed following the demonstration that
DMS treatment is alone sufficient to terminate Taq polymerization
thus obviating the need for the piperidine cleavage step (24). In
the present study it is also clear that a piperidine step is not
required for those agents that alkylate at the guanine-N7 position
to terminate the Taq polymerase. This may be due to the
denaturing incubation at 95 °C which precedes the Taq
polymerase reaction being sufficient to cleave the glycosidic
bonds of alkylated purines (25,26) which would lead to
termination of the enzyme one nucleotide before the damaged
base. Alternatively, the bulky lesions produced by the antitumour
agents could be sufficient to block the progress of the polymerase,
which is presumably the case for agents such as cis-platin which
produce lesions at guanine-N7 which are not converted to strand
breaks by piperidine or heat treatments.
The technique has considerable potential for examining the
sequence selecivity of antitumour agent damage and repair in
single copy gene sequences in cells. Recently a quantitative
polymerase chain reaction based assay has been described for
mapping damage and repair in specific sequences in cells
following UV and 4-nitroquinoline-l-oxide base adduct damage
(27). The assay is based on the quantitative loss (damage) or
recovery (repair) of total counts on agarose gels following
polymerase chain reaction on genomic DNA. The present assay
using denaturing polyacrylamide gels gives the potential to
examine in detail such adduct damage and repair in single copy
genes in cells at the individual base level.
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